Improved procedure for the measurement of telomere length in whole cells by PNA probe and flow cytometry

Objectives: Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo‐PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide. Materials and methods: After telomeric DNA denaturation in hot formamide solution and several washes to remove the ionic solvent, cells were hybridized overnight at room temperature with human telomere‐specific PNA probe conjugated with Cy5 fluorochrome, Cy5‐OO‐(CCCTAA)₃. After stringency washes and staining with ethidium bromide, the cells were analysed by flow cytometry and by using a confocal microscope. Results: Using three continuous cell lines, different in DNA content and telomere length, and resting human peripheral blood T and B lymphocytes, we demonstrated that the oligo‐PNA probe hybridized to telomeric sequences after complete removal of formamide and that in the preserved nucleus, telomeric sequence denaturation is irreversible. Conclusion: According to our experience, oligo‐PNA binding results is efficient, specific and proportional to telomere length. These, our original findings, can form the technological basis of actual in situ hybridization on preserved whole cells.     

Temperature and urea induced denaturation of the TRP‐cage mini protein TC5b: A simulation study consistent with experimental observations

The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20‐residue Trp‐cage mini‐protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force‐field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR‐NOE atom–atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40–90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force‐field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side‐chain side‐chain contact (Trp6‐Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced.     

Effect of amides and ureas on the reversible gelation of arachin

The effect of formamide and urea and their amino-substituted derivatives dimethyl formamide and tetramethyl urea (at 1 m level) on thermal denaturation and protein protein interactions (at pH 3.6) that led to gelation of arachin were studied by gel melting temperature, electrophoresis, u.v. difference and fluorescence spectral measurements. Melting temperature and electrophoretic measurements showed that formamide and urea decreased the heat-induced protein-protein interactions while their methyl derivatives had the opposite effect. Melting temperature measurements also revealed a decrease in both -ΔH_bonding and -ΔS_bonding in the presence of formamide and urea while their methyl derivatives increased these thermodynamic parameters. In both the cases urea and tetramethyl urea had a greater effect on changing both the thermodynamic parameters compared with formamide and dimethyl formamide respectively. U.v. difference and fluorescence spectral measurements suggested that addition of formamide, urea and their methyl derivatives at 1 m level to orachin at pH 3.6 and room temperature induced unfolding. Addition of these compounds to the heated arachin solution at the same pH also promoted the thermal denaturation of the protein. The effectiveness followed the order tetramethyl urea > urea > dimethyl formamide > formamide. The promotive effect of formamide and urea on thermal denaturation and their preventive effect on the protein-protein interactions of arachin could be due to their favourable interaction with interpeptide hydrogen bonds. On the other hand, the promotive effect of dimethyl formamide and tetramethyl urea on the thermal denaturation of the protein may be due to their solubilization effect on the intraprotein hydrophobic interactions. The increase in protein-protein interactions in the presence of these compounds could be due to an increase in interprotein hydrogen bonding. This hypothesis of the mechanism of the additives on the heat-induced protein-protein interactions at pH 3.6 is consistent with the measured thermodynamic parameters of gelation.     

A comparative study of in situ hybridization procedures using cRNA applied to Drosophila hydei salivary gland chromosomes

The effect of different denaturation and hybridization procedures on the efficiency of in situ ³H-cRNA hybridization with DNA in the polytene chromosomes of Drosophila hydei was investigated.Denaturation of the DNA in the squash preparations with 90% formamide in 0.1 × SSC at 65 °C for 2.5 h gave a significantly higher retention of radioactivity following in situ hybridization than did denaturation by 30 sec incubation in boiling 0.1 × SSC.A comparison of the effect of various SSC concentrations in the hybridization mixture revealed that among the SSC concentrations tested, 3 × SSC or 4 × SSC gave the highest efficiency of hybrid formation.Hybridization in 50% formamide at 20 °C resulted in continuing hybrid formation over a period of 3.5 h, the majority of the cRNA/DNA hybrids being formed within the first 10 min of the incubation period. The thermal dissociation profile of in situ cRNA/DNA hybrids formed in 50% formamide, 4 × SSC at 20 °C, as determined in 0.1 × SSC indicated a T_m of 66 °C. The shape of the profile and the results of competition experiments suggested a high fidelity of base-matching in the in situ ³H-cRNA/DNA hybrids.Non-chromosomal background labeling in autoradiographs of polytene chromosomes hybridized with ³H-cRNA was effectively reduced by adding a 200–1000 fold excess of cold 28S + 18S RNA.     

Kinetics of the helix-coil transition in DNA

The kinetics of the helix-coil transition have been investigated for T2 and T7 phage DNA in a formamide-water-salt mixed solvent using a slow temperature perturbation technique (applicable to kinetic processes with rate constants ? 3 min^?1). In this solvent degradation of the DNA is effectively suppressed. Complex kinetic curves are observed by absorbance and viscosity measurements for the response to denaturing perturbations in the transition region. Analysis of the decay curves indicates that the denaturation reaction in this time range can be treated as a first-order reaction with a variable first-order rate parameter, k, the derivative of the logarithm of the absorbance or viscosity change with respect to time. In the approach to denaturation equilibrium in the transition region, the rate parameter is determined only by the instantaneous extent of denaturation of the molecules. Near equilibrium, the rate parameter assumes a constant value characteristic of the equilibrium state. In this region, where the denaturation reaction proceeds as a simple first-order process, both the decay of absorbance (reflected local conformational change) and the decay of solution viscosity (reflecting macromolecular conformational change) are characterized by the same constant value of k. In 83% formamide, 0.3M Na⁺, the rate parameter k for T2 DNA decreases from an extrapolated value of 2.0 min^?1 at 0% denaturation to 0.11 min^?1 at 90% denaturation. Rate parameters determined for T7 DNA at the same counterion concentration and fraction of denaturation are approximately five times as large as those cited for T2 DNA, indicating an inverse proportionality of rate constant to molecular length. On the other hand, simple first-order kinetic responses with constant k are obtained for renaturing perturbations within the transition, indicating that the mechanism of rewinding differs, in most cases, from that of unwinding. Only in the limit of very small perturbations about a given equilibrium position are the rate constants k obtained from denaturing and renaturing perturbations equal. For perturbations of finite size, it appears possible that an intramolecular initiation or nucleation event may precede rewinding and limit the rate of this reaction. The rate parameters again are approximately inversely proportional to molecular weight. The one exception to the first-power dependence on molecular weight appears when temperature jumps are made upward into the post-transition region. Here the molecular-weight dependence is second power, but complications arising from the different strand-separation properties of T2 and T7 DNA's make interpretation difficult. The previously used model of friction-limited unwinding appears to fit all the observations except for the molecular-weight dependence.     

Circular forms of Uukuniemi virion RNA: an electron microscopic study.

Because the ribonucleoprotein forms of the segments of the Uukuniemi virus genome have previously been characterized as circular, we examined the isolated RNAs by electron microscopy under conditions of increasing denaturation. After spreading under moderately denaturing conditions (50 or 60% formamide), 50 to 70% of the molecules were circular. Increasing the formamide concentration to 70 and 85% decreased the number of circular forms, and only linear forms were observed after incubation of the RNA at 60 degrees C for 15 min in 99% formamide. When spread from 4 M urea-80% formamide--another condition known to denature RNA--only 5 to 30% circular molecules were observed. Pretreatment of the RNA with 0.5 M glyoxal at 37 degrees C for 15 min prior to spreading from 50% formamide gave less than 5% cirucular forms. Length measurement of the molecules showed that they were not significantly degraded by any of the methods employed. The circular molecules were destroyed by treatment with pancreatic RNase, but were unaffected by DNase or proteinase K treatment. After complete denaturation of the RNA, the circles could be reformed under reannealing conditions. We conclude that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3'' and 5'' ends of linear molecules.     

Vicia faba chloroplast DNA has only one set of ribosomal RNA genes as shown by partial denaturation mapping and R-loop analysis

Summary Broad-bean (Vicia faba) chloroplast DNA (cpDNA) was isolated and characterized. The intact DNA is circular and has a molecular weight of 79.8x 10⁶ dalton. Electron microscopic analysis of self-annealed intact single-strand circles show that it does not have a large double-stranded inverse repeat as seen in spinach chloroplast DNA. Only one ribosomal RNA gene (one set of 16S and 23S rRNA sequences) was found in preparations of R-loops between the Vicia rRNA and cpDNA circles. A restriction enzyme map for SalI and KpnI was derived by comparing the partial denaturation pattern of the fragments with the pattern of the intact circle. The map was confirmed by gel analysis. The ribosomal RNA gene was localized on the SalI fragment 3b by R-loop analysis. SalI fragment 1a although it contains a G-C rich region did not form R-loops with rRNA. Partial denaturation patterns of spinach cpDNA circles and BglI fragments were determined and from this the position of the fragments mapped. This confirmed the reliability of these methods for the arrangement of restriction enzyme fragments along circular molecules. The structures of the two cpDNAs were compared.     

Electron Microscopy of Supercoiled pEJ4 DNA Containing Homopurine · Homopyrimidine Sequences

Abstract

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine · homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine · homopyridine tract assumes an unusual conformation) a single thick “stem” or a “denaturation bubble” in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC 19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The “denaturation bubbles” were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only “stems”. The “stems” were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine · homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.     

Localisation of reiterated nucleotide sequences in Drosophila and mouse by in situ hybridisation of complementary RNA

The location of highly reiterated nucleotide sequences on the chromosomes has been studied by the technique of in situ hybridisation between the DNA of either Drosophila melanogaster salivary gland chromosomes or mouse chromosomes and tritium labelled complementary RNA (c-RNA) transcribed in vitro from appropriate templates with the aid of DNA dependent RNA polymerase extracted from Micrococcus lysodeikticus. The location of the hybrid material was identified by autoradiography after RNase treatment. — When Drosophila c-RNA, transcribed from whole DNA, was annealed with homologous salivary chromosomes in the presence of formamide the well defined labelling was confined to the chromocentre. With heat instead of formamide denaturation there was evidence of discontinuous labelling in various chromosome regions as well, apparently associated with banding. Xenopus ribosomal RNA showed no evidence of annealing to Drosophila chromosomes with the comparatively short exposure times used here. — When mouse satellite DNA was used as template the resulting c-RNA showed no hybridisation to Drosophila chromosomes but, when annealed with mouse chromosomes, the centromeric regions were intensely labelled. The interphase nuclei showed several distinct regions of high activity which suggested aggregation of centromeric regions of both homologous and non-homologous chromosomes. The results of annealing either c-RNA or labelled satellite DNA to homologous chromosomes were virtually indistinguishable. Incubation of Drosophila c-RNA with mouse chromosomes provided no evidence of localisation of grains. — It is inferred that both in mouse and Drosophila the centromeric regions of all chromosomes are enriched in highly reiterated sequences. This may be a general phenomenon and it might be tentatively suggested that the highly reiterated sequences play some role in promoting the close physical approximation of homologous and non-homologous chromosomes or chromosome regions to facilitate regulation of function.     

An enhanced thermostability in thermophilic 5-S ribonucleic acids under physiological salt conditions.

The secondary structure of 5-S rRNAs of Thermus aquaticus (an extreme thermophile), Bacillus stearothermophilus (a moderate thermophile) and Escherichia coli (a mesophile) was compared using thermal denaturation techniques under varying ionic conditions. At a low ionic strength (10 mM K+), the Tm of T. aquaticus 5-S RNA differed by only 1 degrees C from that of E. coli RNA and the molecule was fully denatured well below the optimum growth temperature of the thermophile. The internal Na+, K+ and Mg2+ concentrations of T. aquaticus cells were determined to be 91 mM, 130 mM and 59 mM, respectively. Under these salt conditions, T. aquaticus 5-S RNA was significantly more stable than E. coli RNA and the 5-S RNA from B. stearothermophilus was intermediate as is its optimum growth temperature. The results suggest that the thermostability of macromolecules from thermophilic organisms may be specially dependent on the internal salt concentration. Furthermore, under these salt conditions, most of the secondary structure of the RNA remained stable at the optimum growth temperatures suggesting that ribosomal RNAs of thermophilic organisms contribute more to the thermostability of the ribosome than previously thought.     

Formamide denaturation of chromosomal DNA for in situ hybridization and C-band preparation in the guinea pig, Cavia porcellus

Cytological preparations were incubated in 0.07 N NaOH at room temperature or 90% formamide (final salt concentration 2 × SSC) at either 65 °C or 37 °C for 2.5 h to denature guinea pig chromosomes. Chromosomes treated with NaOH or formamide at 65 °C showed a large amount of DNA loss, while chromosomes treated with formamide at 37 °C showed little or no DNA loss. Repeated sequences were isolated from guinea pig DNA and [³H]cRNA was transcribed with Escherichia coli RNA polymerase for in situ hybridization. Localization of the [³H]cRNA occurred in the centromeric regions and C-band positive short arms of almost all of the chromosomes in the NaOH preparations. Chromosomes treated with formamide at 65 °C showed the same grain distribution with a decrease in the number of grains/cluster. Slides incubated in formamide at 37 °C showed localization in only a few chromosomes and the number of grains/cluster was greatly diminished. Thermal denaturation of isolated chromatin indicated that incubation of chromosomes in formamide at 37 °C did not fully denature the DNA. C-bands could be induced by treating slides in formamide at either 65 °C or 37 °C when followed by a “reassociation” in 2 × SSC at 65 °C for 16 h. If the “reassociation” step was omitted, C-bands were found in the 65 °C formamide slides but not the 37 °C formamide slides.     

Location of DNA ends in P2, 186, P4 and lambda bacteriophage heads

When mature phage particles were suspended in a solution containing formaldehyde (0.07 m-Na⁺, pH 9.0, 10% HCHO for 10 min at 23 °C) and the mixture then spread for electron microscopy in the presence of 50% formamide and cytochrome c, the phage lysed and a high proportion of the DNA molecules were seen to be attached to phage tails. The phage tails were found to be attached at only one end of each DNA molecule and denaturation mapping showed that this end was unique for each of the phages P2, 186, P4 and λ. It is argued that in these mature phage particles one specific end of the DNA molecule is present at the head-tail attachment site.     

Effect of organic solvents on the properties of the complexes of DNA with proflavine and similar compounds

In order to obtain information on the binding forces involved in the formation of the complex proflavine–DNA by the stronger process I, the stability of the complexes was investigated in the presence of various organic solvents, methanol, ethanol, n-propanol, isopropanol, formamide, dimethyl sulfoxide, p-dioxane, glycerol, and ethylene glycol. Quantitative data on binding in terms of K/n and r were obtained by means of absorption and fluorescence spectra, as well as by a thermal denaturation technique. All organic solvents used decrease the binding ability of the dye. The effectiveness of the solvents increases with their hydrocarbon content, but can hardly be related to their dielectric constant. The complex formation is effectively suppressed by organic solvent concentrations, in which DNA still preserves its double-helical conformation. These results demonstrate the importance of hydrophobic forces in the formation of the complex proflavine–DNA in aqueous solution. The similarity in spectroscopic properties of proflavine bound to DNA by process I and the same dye dissolved in an organic solvent make it possible to interpret the observed red shift of the long-wavelength absorption peak as being due to the interaction of the dye molecules with the less polar environment. The same behavior was found for other dyes capable of intercalation like purified trypaflavine, phenosafranine and ethidium bromide. However, intercalation is not a necessary condition, as it was shown in the case of pinacyanol, which binds only at the surface of DNA.     

Amyloid Fibrillation of Insulin under Water-Limited Conditions

Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation.     

Size and structure of the genome of infectious pancreatic necrosis virus.

The genome of infectious pancreatic necrosis virus consists of two segments of dsRNA, in equimolar amounts, with molecular weights of 2.5 X 10(6) and 2.3 X 10(6) daltons, as determined by polyacrylamide gel electrophoresis and autoradiography. The viral RNA was resistant to ribonuclease, and in sucrose gradient it co-sedimented at 14S with RNase resistant RNA from virus infected cells. Upon denaturation in 98% formamide, the viral genome sedi-mented at 24S in formamide sucrose gradient and became sensitive to RNase. Denatured 24S viral RNA did revert to its undenatured 14S form upon recentrifugation in aquaeous sucrose gradient (0.1 M NaCL), but co-sedimented with the denatured large size class of reovirus 25S RNA. The same results were obtained if the native viral RNA was pre-treated with ribonuclease before denaturation, indicating the absence of exposed single strainded regions in the viral genome. Since infectious pancreatic necrosis virus contains only two dsRNA segments it does not belong to the family Reoviridae and may represent a new group of viruses.     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

Studies on the biosynthesis of TMV

Summary Heating of the TMV replicative form (RF) above a certain temperature (T _m)causes a sharp shift from RNase resistance to sensitivity. The T _mwas determined at different salt concentrations and in the presence of formamide.The kinetics of the annealing reaction between TMV RNA and its complementary RNA was studied, and the rate constant was estimated. Under the chosen conditions which are appropriate for annealing, no dissociation of double-stranded TMV RNA was detected. The kinetic data permitted a maximum estimate of the equilibrium constant of the annealing (or dissociation) reaction.     

Raman spectral studies of nucleic acids. XI. Conformations of yeast tRNAPhe and E. coli ribosomal RNA in aqueous solution and in the solid state

Laser-excited Raman spectra of tRNA^Phe from yeast and of fractionated 16S and 23S rRNA from E. coli are reported for samples in aqueous solution and in the solid state. The Raman scattering spectrum of each RNA is not significantly altered by the change from an aqueous to a solid environment and displays the same characteristic frequencies and intensities associated with ordered polyribonucleotide structures. Unlike DNA, the backbone conformation of RNA thus appears to be largely insensitive to gross changes in the degree of hydration. Raman scattering from the phosphate group vibrations of aqueous tRNA_yeast^Phe is qualitatively and quantitatively the same as obtained from previously studied tRNA's and is indicative of a highly ordered conformational structure in which some 85% of the nucleotide residues are in ordered configurations. The major differences observed between spectra of tRNA and rRNA are attributed to differences in base composition of these RNA's.     

Amyloid Fibrillation of Insulin under Water-Limited Conditions

Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation.